Pyrolysis mass spectrometry of coastal Bermudagrass (Cynodon dactylon (L.) Pers.) and ‘Kentucky-31’ tall fescue (Festuca arundinacea Schreb.) cell walls and their residues after ozonolysis and base hydrolysis

Animal Feed Science and Technology, 32 ( 1991 ) 17-26 Elsevier Science Publishers B.V., Amsterdam

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Pyrolysis mass spectrometry of coastal Bermudagrass (Cynodon dactylon (L.) Pers. ) and 'Kentucky-3 l' tall fescue (Festuca arundinacea Schreb. ) cell walls and their residues after ozonolysis and base hydrolysis W. Herbert Morrison, III L*, Martin A. Scheijen 2 and Jaap J. Boon 2 LUSDA/ARS R.B. RussellAgriculturaIResearch Center, Plant Structure and Composition Research Unit, P.O. Box 5677, Athens, GA 30613 (U.S.A.) "-Unit for Mass Spectrometry of Macromolecular Systems, FOM Institute for Atomic and Molecular Physics, Kruislaan 407. 1098 SJ Amsterdam (The Netherlands)

ABSTRACT Morrison, III, W.H., Scheijen, M.A. and Boon, J.J., 1991. Pyrolysis mass spectrometry of coastal Bermudagrass (Cynodon dactylon (L.) Pers. ) and 'Kentucky-31' tall fescue (Festuca arundinacea Schreb. ) cell walls and their residues after ozonolysis and base hydrolysis. Anim. FeedSci. Technol., 32:17-26. Cell walls from coastal Bermudagrass (Cynodon dactylon (L.) Pets.) (CBG) and 'Kentucky-31' tall fescue (Festuca arundinacea Schreb.) (K-31) were treated with ozone and the resulting residue with sodium hydroxide. The aromatics and polysaccharides of the treated cell walls and the residues from ozone and ozone/base treatments were studied by pyrolysis mass spectrometry. Ozone effectively removed lignin from the cell walls of both grass species. The residue from ozone-treated CBG cell walls showed mass markers characteristic of condensed lignin whereas the base-treated residue from ozone-treated K-31 cell walls showed no lignin remaining in the sample and principally polysaccharides being left. This difference may be due to the type of lignin present in the two grasses which, in turn, may relate to CBG cell walls being less digestible by rumen microorganisms.

INTRODUCTION

Ozone has been used to pretreat agricultural waste products in order to improve their utilization by ruminants (Ben-Ghedalia and Miron, 1981; BenGhedalia, 1982; Ben-Ghedalia and Shefet, 1983; Ben-Ghedalia et al., 1983; Byrant et al., 1984). Cell wall polysaccharides are made more available and in vitro digestion of the by-products is increased (Ben-Ghedalia and Shefet, *Collaborator via Fellowship under OECD Project on Food Production and Preservation.

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W.H. MORRISON, 111ET AL.

1983). Shefet and Ben-Ghedalia (1982) reported a lag of 12-24 h in the digestion of straws which had been treated with ozone and suggested the possibility of a toxic component being formed during ozone treatment. Others have shown that this lag is eliminated if the ozone-treated material is washed with water prior to digestion (Narasimhalu et al., 1989). In earlier work (Morrison and Akin, 1990), we reported on the water-soluble products produced on treatment of coastal Bermudagrass (Cynodon dactylon (L.) Pers. ) (CBG) and 'Kentucky-31' tall fescue (Festuca arundinacea Schreb. ) (K-31 ) whole grass and cell walls with ozone. Among the compounds produced were p-hydroxybenzaldehyde and vanillin which Borneman et al. (1986) reported to be especially toxic and to inhibit the growth and plant cell wall degrading activity of pure and mixed populations of bacteria, which could account for the observed lag. Little work has been reported on the phenolics remaining in the residue from ozone treatment. Pyrolysis mass spectrometry has been used to investigate a number of agricultural products and was reviewed recently (Boon, 1989). Briefly, the sample ( 10-50/zg) is placed on a ferromagnetic wire which is then placed in the mass spectrometer. A magnetic field is generated which raises the temperature of the wire to its Curie point in less than 0.1 s to instantly pyrolyze or vaporize the sample. A second approach is to place the sample on a wire which can be resistively heated at various rates. In this case the wire can be positioned directly in the ion source so that the loss of chemical information owing to inefficient transfer of the pyrolysate to the ion source is minimized. Pyrolysis mass spectrometry (PYMS) produces information on the products arising from evaporation and/or thermal decomposition of the material under study (Boon, 1989 ). These products can provide information on the original matrix which contained these compounds. This study will report on the use of PYMS to investigate phenolics in CBG and K-31 tall fescue cell walls which had been treated with ozone. MATERIALS AND METHODS

Field-grown coastal Bermudagrass ( Cynodon dactylon (L.) Pers. ) (CBG) samples were taken from 6-week-old regrowth on plots near Tifton, GA. 'Kentucky-31' tall fescue (Festucaarundinacea Schreb.) (K-31 ) samples were harvested from 4-6-week-old regrowth on well managed and fertilized fields near Eatonton, GA. All plants were frozen quickly after harvesting and maintained at - 10 °C until experiments were conducted.

Preparation and ozonolysis of cell wall material Samples were prepared from freeze-dried material which had been ground to pass through a 1-mm (20 mesh) screen. Cell wall material was prepared by

PYMS STUDY OF PHENOLICS IN OZONE-TREATED CELL WALLS

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extracting the ground material in Soxhlet apparatus with benzene/ethanol ( 70: 30 v / v ). After drying, the material was treated with a-amylase (Type 6A, Sigma No. A6880). After washing with water, the material was treated with neutral detergent solution (van Soest and Wine, 1967) without the addition of sodium sulfite and dekalin (Hartley et al., 1974). The resulting cell walls were washed with water, acetone and diethylether. The walls were then dried in a vacuum oven. Cell walls were adjusted to 50% moisture by the addition of an equal weight of water. Samples were sealed and equilibrated overnight at 1 ° C and then stored at - 20 ° C until use. All samples were equilibrated to room temperature before weighing. Dry matter ( D M ) analyses were determined by drying moistened and ground samples in a vacuum oven at 60 ° C for 6 h. Ozonolysis was carried out for 15 min using a Welsbach model T-408 ozone generator (Welsbach Ozone Systems, Sunnyvale, CA) as described earlier (Akin and Morrison, 1988 ). There was no attempt to maximize reaction conditions. The ozone-treated samples were stirred with distilled water for 20 rain, then washed with acetone and diethylether. Samples were then placed in a vacuum oven at room temperature to remove the last traces of solvent.

Base treatment Ozonized cell walls (100 mg) were stirred overnight with 5 ml of 1 M NaOH. Cell walls were filtered through a coarse sinter, washed with water, acetone and diethylether and dried as described above.

Sample preparation for PYMS A suspension of the cell walls was prepared by grinding 1-2 mg in a small glass vial with 2 drops of water. An aliquot of the sample ( 10-50 gg) was transferred to the heating wire and the water evaporated in vacuo.

Curie poin t PYMS Curie point PYMS was performed on the FOMautoPYMS system described previously (Boon et al., 1984). Conditions of pyrolysis were: Curie temperature, 510 ° C; heated pyrolysis chamber, 160 ° C; temperature rise time, 0.1 s; total heating time, 0.8 s; expansion chamber temperature, 200°C; electron impact energy, 15 eV; mass range, 25-220; scan speed, 10 scans s- ~;total number of scans averaged 200. Samples were analyzed in quadruplicate.

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W.H. MORRISON,III ET AL.

Desorption electron impact mass spectrometry (DEIMS) Desorption electron impact mass spectrometry (DEIMS) was performed on a JEOL DX 303 double focussing (E/B) mass spectrometer equipped with a combination EI/CI ion source (Pouwels and Boon, 1990). The sample was applied to a platinum wire of a direct insertion probe. In-source pyrolysis was achieved by a programmed heating current from 0 to 0.8 A at a rate of I A min-L Magnetic scans were recorded under the following conditions: accelerating voltage 2.2 kV; mass range, m/z 20-1000; cycle time, 1 s; electron impact energy, 16.0 eV; source temperature, 180°C; source pressure, l0 -6 Torr; post-acceleration voltage, 5 kV. The spectra were recorded and processed using a JEOL DA 5000 computer system.

Multivariate analysis The pyrolysis mass spectra are normalized by expressing the mass intensities as a percentage of the total ion count. Factor discriminant analysis was performed on the PYMS data file using a modified ARTHUR package (Infometrix, Seattle, WA) especially adapted to accept MS data (Hoogerbrugge et al., 1983; Boon et al., 1984). A discussion of its use with pyrolysis mass spectral data can be found in Scheijen et al. (1989). RESULTS AND DISCUSSION

Figure 1 shows typical Curie point pyrolysis low-voltage E1 mass spectra of CBG and K-31 walls and the residues after treatment with ozone followed by treatment with 1 M NaOH. There are noticeable differences between the original cell walls and those treated with ozone. Markers for phenolic compounds (m/z 120, 124, 137, 138, 150, 152, 154, 164, 178, 180, 182, 194, 196, 208, 210) which are evident in the original samples are greatly reduced or absent in the ozone-treated sample. The 4-vinylphenol, marked by m/z 120, and 4vinylguaiacol marked by m/z 150, are indicative ofp-coumaric and ferulic acid esters, respectively (Boon et al., 1982 ). The other markers are indicative of a mixed guaiacyl-syringyl lignin (Boon et al., 1987), although the percentage of syringl units is relatively low (peaks m/z 154, 182, 194, 210). This would be expected from the high percentage of leaf in the sample and the age of the forage when harvested. This supports earlier work (Morrison and Akin, 1989 ) in which the water-soluble oxidation products ofp-coumaric and ferulic acid are removed when the cell walls are washed with water after ozone treatment. Some of these compounds have been shown to be toxic to rumen microorganisms (Borneman et al., 1986) and simply washing the substrate with water eliminates the lag in digestion which occurs in unwashed ozonetreated samples (Narasimhalu et al., 1989 ).

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Although a selective preferential attack by ozone on carbon-carbon double bonds is expected to occur (Kolsaker and Bailey, 1967 ), our data show that all lignin-derived mass peaks are drastically reduced with respect to the PYMS data from the polysaccharides in the sample. The spectrum of the residue from ozone-treated K-31 is an almost pure polysaccharide spectrum. Earlier work has shown that cell wall sugars are much more available for rumcn

22

W.H. MORRISON, III ET AL.

digestion after forage material is treated with ozone. Electron micrographs of leaf sections which have been subjected to rumen microorganisms before and after ozone treatment show much greater breakdown of the cell wall structure for K-31 than CBG. Apparently little structural lignin remains after ozone treatment of K-31 compared with CBG as shown in Fig. l E and F. The polysaccharides are the predominant feature in K-31 (Fig. 1F) and account for its greater digestibility. Base treatment is expected to remove hemicellulose. Indeed a strong reduction in the relative intensity m / z I 14, indicative of pentosans, xylans and arabinoxylans (Ohnishi et al., 1977), is observed in Fig. 1E and F. The CBG spectrum in Fig. l E shows a series oflignin marker peaks with higher relative intensities than in Fig. IC. These relative mass intensities point to the original guaiacyl-syringyl lignin with the exception of m/z 120 from p-coumaryl units which is substantially lower than in Fig. IA. The K-31 spectrum in Fig. IF does not show any sign of increased intensities of lignin marker peaks, which points to a complete removal of phenolic compounds as a result of the ozone treatment. These differences are graphically demonstrated in Fig. 2 showing a discriminant function score map of the first and second discriminant function of the PYMS data. The geometric distance on the map is a measure of the relative difference in sample composition. The mass spectra presented in this figure are discriminant function spectra of Dl and D2 graphically rotated over about 45 ° in order to accentuate the mass peaks discriminating the various samples. The untreated cell walls of CBG and K-31 are both clearly characterized by m/z 120 and 150 and the other ether lignin peaks (Fig. 2A). Ozone treatment leads to the removal of these markers and consequently to PYMS spectra with an increased intensity of polysaccharide markers by pentose-dominatedhemicellulose in the upper quadrant D l + D2 + (Fig. 2B ) and by mainly hexosan (cellulose) in the lower quadrant D1 + D 2 - (Fig. 2D). The presence of a residual lignin in the CBG sample remaining after treatment with ozone and base is exemplified by its relative position in D 1- D 2 - and its discriminating mass peaks (Fig. 2C). Further evidence for the fate oflignin during ozonization was obtained from the DEIMS data. Figure 3 gives the time-integrated low voltage DEIMS data from the untreated CBG and K-31 samples and their ozone-base treated residues. As pyrolysis in the ion source gives a better transmission of higher too-

Fig. 2. Map resulting from discriminant analysis of PYMS fingerprints of the CBG and K-31 cell walls and their residues after treatment with ozone and subsequent base treatment. CBG, open symbols; K-3 l, closed symbols, o, untreated cell walls;/x, ozone-treated; l"I, ozone/basetreated. ( A ) - ( D ) show reconstructed mass spectra of the first discriminant function after rotating D~ over 134, 34, 214 and 314 °, respectively.

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lecular weight compounds, we chose to plot the mass range from m/z 110 to 420. The relative intensities of the mass peaks in the lower mass range are fairly similar to the Curie point PYMS data from the FOMautoPYMS although m/z 210 is clearly more prominent in the DEIMS data. The latter phenomenon points to a lower sensitivity at the higher masses in the FOMautoPYMS. The mass peaks from m/z 250 to 420 are indicative of condensed phenolic compounds. The mass peak profile is similar to that reported for barley straw (Boon, 1989). The relative intensities of these peaks in the spectra of K-31 and CBG indicate that the lignins in these two grasses are qualitatively different. The presence of homologous series with mass shifts of 30 u (e.g. 242, 272, 302, 298, 328, 358, 388, 418; 310, 340, 370, 400) points to structural regularities in the condensed products of p-coumaryl, guaiacyl and syringyl units (Hempfling and Schulten, 1988 ). Ozone treatment and the subsequent basic hydrolysis of CBG affects mainly the relative intensity of many of these characteristics, suggesting that some lignin remains intact (Fig. 3 ). In the case of K-31, little evidence remains for any lignin after these treatments however, which confirms the earlier observations. The high molecular weight lignin markers found in CBG suggest a core lignin present in CBG that is very low or not present in K-31. Ben-Ghedalia (1982) reported that although ozonolysis of cottonstraw resulted in a marked degradation of the lignin, there was a residue of undegraded core lignin that was not affected by ozone treatment. Although the lignin profile of CBG is affected by ozone and base treatment, both Curie point PYMS and in-source MS point to a condensed lignin remaining in the polysaccharide-rich residue. This could well account for the differences in digestion mentioned above and a difference in the lignin-carbohydrate backbone. Further work is needed to isolate this polyphenolic material free of polysaccharide for further characterization.

REFERENCES

Akin, D.E. and Morrison, lIl, W.H., 1988. Ozone treatment of forage: Structure and digestibility of different lignified cell walls. Crop Sci., 28: 337-342. Ben-Ghedalia, D., 1982. Degradation of cell-wall monosaccharides of chemically treated grape branches by rumen microorganisms. Eur. J. Appi. Microbiol. Biotechnoi., 16: 142-145. Ben-Ghedalia, D. and Miron, J.J., 1981. Effects of sodium hydroxide, ozone and sulphur dioxide on the composition and in-vitro digestibility of wheat straw. J. Sci. Food Agric., 32: 224228. Ben-Ghedalia, D. and Shefet, G., 1983. Chemical treatment for increasing the digestibility of cotton straw. II. J. Agric. Sci., 100: 401-406.

26

W.H. MORRISON,I11El"AL.

Ben-Ghedalia, D., Shefet, G. and Dror, Y., 1983, Chemical treatment for increasing the digestibility of cotton straw. I. J. Agric. Sci., 100: 393-400. Boon, J.J., 1989. An introduction to pyrolysis (gas chromatography) mass spectrometry oflignocellulose material with case studies on barley straw var. OECD, corn stem var. BM, eta I PHO and LglI and Agropyron species. In: A. Chesson and E.R. (~rskov (Editors), Physicochemical Characterization of Plant Residues for Industrial and Feed Use. Elsevier Applied Science, Barking, pp. 25-49. Boon, J.J., Wetzel, R.G. and Godschalk, G.L., 1982. Pyrolysis mass spectrometry of some Scirus species and their decomposition products. Limnol. Oceanogr., 27: 839-848. Boon, J.J., Tom, A., Brandt, B., Eijkel, G.B., Kistemaker, P.G., Notten, F.J.W. and Mikx, F.H.M., 1984. Mass spectrometry and factor analysis of complex organic matter from bacterial culture environment of Bacteroides gingivalis. Anal. Chim. Acta, 163:193-205. Boon, J.J., Pouwels, A.D. and Eijkel, G.B., 1987. Pyrolysis high resolution gas chromatography mass spectrometry studies of a beech lignin fraction. Biochem. Soc. Trans., 15:170-174. Borneman, W.S., Akin, D.E. and van Eseltine, W.P., 1986. Effects of phenolic monomers on rumen bacteria. Appl. Environ. Microbiol., 52:1331-1339. Bryant, F.C., Mills, T., Pitts, J.S., Carrigan, M. and Wiggers, E.P., 1984. Ozone treated mesquite for supplementing steers in west Texas. J. Range Mange., 37: 420-422. Hartley, R.D., Jones, E.C. and Fenlon, J.S., 1974. Prediction of the digestibility of forages by treatment of their cell walls with cellulolytic enzymes. J. Sci. Food Agric., 25: 947-954. Hempfling, R. and Schulten, H.-R., 1988. Characterization and dynamics of organic compounds in forest humus studied by pyrolysis-gas chromatography-electron impact mass spectrometry and pyrolysis-high-resolution field ionization mass spectrometry. J. Anal. Appl. Pyrolysis, 13:319-326. Hoogerbrugge, R., Willig, R.J. and Kistermaker, P.G., 1983. Discriminant analysis by double stage principal component analysis. Anal. Chem., 55:1711-1712. Kolsaker, P. and Bailey, P.S., 1967. Ozonation of compounds of the type ArCHCHG; ozonation in methanol. Acta Chem. Scand., 21: 537-546. Morrison, W.H. and Akin, D.E., 1990. Water soluble products from ozonolysis of grasses. J. Agric. Food Chem., 38: 678-681. Narasimhalu, P., Graham, H. and Aman, P., 1989. Effects of ozone treatment on the chemical composition and in-vitro degradation of triticale internodes and red clover stems. Anim. Feed Sci. Technol., 24: 159-172. Ohnishi, A., Kato, K. and Tagaki, E., 1977. Pyrolytic formation of 3-hydroxy-2-1actone from xylan, xylo-oligosaccharides and methyl xylopyranosides. Carbohydr. Res., 58: 387-395. Pouwels, A.D. and Boon J.J., 1990. Analysis of beech wood samples, its milled wood lignin and polysaccharide fraction by platinum filament pyrolysis mass spectrometry. J. Appl. Anal. Pyrolysis, 17: 97-126. Scheijen, M.A., Boer, B.B., Boon, J.J., Hass, W. and Heemann, V., 1989. Evaluation of a tobacco fractionation procedure by pyrolysis mass spectrometry combined with multivariate analysis. Beitr. Tabakforsch. Int., 14, 261-282. Shefet, G. and Ben-Ghedalia, D., 1982. Effects of ozone and sodium hydroxide treatments on the degradability of cotton straw monosaccharides by rumen micororganisms. Eur. J. Appl. Microbiol. Biotechnol., 15:47-51. Van Soest, P.J. and Wine, R.H., 1967. Use of detergents in the analysis of fibrous feeds. IV Determination of plant cell wall constituents. J. Assoc. Off. Agric. Chem., 50:50-55.